Mutational analysis of RsrA, a zinc-binding anti-sigma factor with a thiol-disulphide redox switch

In the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression o f the thioredoxin system is modulated by a sigma factor called sigma (R) in response to changes in the cytoplasmic thiol-disulphide status, and the ac tivity of sigma (R) is controlled post-translationally by an anti-sigma fac tor, RsrA. In vitro, the anti-sigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol-disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and sig ma (R)-dependent transcription, confirming that RsrA is a negative regulato r of sigma (R) and a key sensor of thiol-disulphide status. Targeted mutage nesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti-sigma factor activity and that a mutant RsrA prote in containing only these three cysteines was active and still redox sensiti ve in vivo. We also show that RsrA is a metalloprotein, containing near-sto ichiometric amounts of zinc. On the basis of these data, we propose that a thiol-disulphide redox switch is formed between two of C11, C41 and C44, an d that all three residues play an essential role in anti-sigma factor activ ity in their reduced state, perhaps by acting as ligands for zinc. Unexpect edly, rsrA null mutants were blocked in sporulation, probably as a conseque nce of an increase in the level of free sigma (R).